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WO2008108964A2 - Protéine kinase 1 associée à l'apoptose (dapk1) et ses utilisations pour le traitement de la leucémie lymphocytique chronique - Google Patents

Protéine kinase 1 associée à l'apoptose (dapk1) et ses utilisations pour le traitement de la leucémie lymphocytique chronique Download PDF

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WO2008108964A2
WO2008108964A2 PCT/US2008/002625 US2008002625W WO2008108964A2 WO 2008108964 A2 WO2008108964 A2 WO 2008108964A2 US 2008002625 W US2008002625 W US 2008002625W WO 2008108964 A2 WO2008108964 A2 WO 2008108964A2
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dapkl
cll
expression
cells
chronic lymphocytic
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WO2008108964A3 (fr
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Christoph Plass
Albert De La Chapelle
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The Ohio State University Research Foundation
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Priority to US12/529,214 priority Critical patent/US20100184037A1/en
Priority to EP08726202A priority patent/EP2126129A4/fr
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Publication of WO2008108964A3 publication Critical patent/WO2008108964A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention is directed to certain novel compounds, methods for producing them and methods for treating or ameliorating a kinase mediated disorder, and in particular to death associated protein kinases 1 (DAPKl). More particularly, this invention is directed to compounds useful as selective kinase inhibitors, methods for producing such compounds and methods for treating or ameliorating chronic lymphocytic leukemia (CLL).
  • DAPKl death associated protein kinases 1
  • CLL Chronic lymphocytic leukemia
  • a method for determining susceptibility to chronic lymphocytic leukemia in a subject includes determining a loss or reduced expression of death associated protein kinase 1 (DAPKl) .
  • DAPKl death associated protein kinase 1
  • a method for determining susceptibility to chronic lymphocytic leukemia in a subject by determining the occurrence of reduced DAPKl expression in combination with frequent promoter methylation in the CLL cells.
  • the DAPKl silencing is due to modulation of upstream signal, where at least one signal comprises HOXB7, a homeobox containing transcription factor mediating a variety of developmental processes, including hematopoietic differentiation and lymphoid development.
  • composition that includes a single-nucleotide germline mutation (c.l-6531A>G) [SEQ ID NO: 1] upstream of DAPKl, which segregates with a CLL phenotype.
  • a tumor suppressor for chronic lymphocytic leukemia includes one or more epigenetic silencing and/or mutations in death associated protein kinase 1 (DAPKl).
  • composition that includes at least one mutation in the homeobox containing transcription factor binding site for HOXB7, which is a binding site in DAPKl regulatory region and/or promoter methylation which results in significant reduction in the expression of the pro-apoptotic gene DAPKI in familial cases of CLL and in sporadic CLL.
  • a DAPKI promoter construct #2 (c.1 - 1545 to c.l-1151bp) [SEQ ID NO: 2], covering bisulfite region Al (displaying extensive promoter methylation).
  • a CLL specific SNP c.1-6531A>G .
  • a 357 bp fragment including C.16531G (DAP-G) [SEQ ID NO: 4] ligated upstream into luciferase construct #1 containing a DAPKI promoter is provided.
  • a method for determining susceptibility to chronic lymphocytic leukemia that includes integrating genetic and epigenetic data for the discovery of predisposing genes in cancer.
  • Also provided is a method for determining susceptibility to chronic lymphocytic leukemia that includes combining linkage analysis and epigenetic studies using at least the down regulation of DAPKl .
  • One method for determining susceptibility to chronic lymphocytic leukemia includes screening of familial CLL cases for DAPKI promoter methylation.
  • the determination of susceptibility to chronic lymphocytic leukemia can include identifying at least one DNA methylation event that is reversible for the development of novel treatment regimens in CLL involving epigenetic therapies for gene reactivation.
  • the method can include: detecting a level of expression of a marker selected from a group of markers associated with CLL in a test sample from the human subject; and detecting the level of expression of the marker in a control sample from normal tissue from the human subject, wherein the level of expression of the marker in the control sample differs from the level of expression of the marker in the test sample when the subject is afflicted with CLL, and wherein the marker is encoded by a gene.
  • the test sample from the subject can be cells obtained from the subject, such as for example, cells obtained from blood.
  • the levels of expression of the marker in the control sample and in the test sample are assessed by a method comprising: contacting a first array of probes with a first population of nucleic acids derived from one or more cells from the test sample; contacting a second array of probes with a second population of nucleic acids derived from one or more cells from the control sample; and determining relative hybridization of the first array of probes to the first population of nucleic acids relative to hybridization of the second array of probes to the second population of nucleic acids.
  • the first and second population of nucleic acids can be RNA and/or DNA.
  • the first population of nucleic acids can be amplified prior to contacting to the first array of probes or the second population of nucleic acids is amplified prior to contacting the second array of probes.
  • the marker can be a nucleic acid, such as RNA or DNA.
  • One or more nucleic acids can be amplified prior to assessing the sample.
  • a method for monitoring the progression of CLL in a human subject includes: detecting in a first sample obtained from the human subject at a first point in time, a level of expression of a marker selected from a group of markers associated with CLL; detecting in a subsequent sample obtained from the human subject at a subsequent point in time, the level of expression of the marker, and comparing the level of expression detected in the first and subsequent detecting samples in order to monitor the progression of CLL, wherein the marker is encoded by a gene selected as described herein.
  • the first and the subsequent samples can be cells obtained from the subject, such as, for example, blood cells.
  • the control sample from the subject can comprise cells obtained from the subject.
  • a method of preventing recurrence of a CLL that comprises administering an effective amount of a composition comprising an effective inhibitor of DAPKl .
  • the method is useful as a therapeutic tool to prevent recurrence or further tumor spread.
  • composition for affecting CLL comprising a DAPKl regulator.
  • an animal model for examining CLL models that comprises administering one or more DAPKl regulators. Also, provided is a method for inhibiting CLL cell migration comprising administering an effective amount of a composition comprising one or more DAPKl inhibitors.
  • a method for treating a patient in need thereof comprising administering a therapeutically effective amount of a pharmaceutical composition which comprises a substance that regulates the activity of DAPKl, as an active ingredient.
  • FIGURES IA and IB shows an analysis of DAPKl promoter methylation by MassARRAY analysis in CLL samples:
  • FIGURE IA - Upper panel shows a schematic representation of the DAPKl gene showing the location of CpG island (black bar) and the four bisulfite reactions amplicons (Al to A4). Amplicons Al to A4 cover 34, 39, 28 and 35 CpGs, respectively and extend from c.1-1573 to c.l -239bps . An arrow indicates the predicted transcription start site.
  • the lower panel shows a heat map of quantitative methylation data for the DAPKl promoter region. Each line represents a sample and each square an analyzed CpG dinucleotide. Methylation frequencies extend from light yellow (0%) to dark blue (100%). Gray indicates no available data.
  • FIGURES 2A - 2E show epigenetic silencing of DAPKl in CLL:
  • FIGURE 2A - pGL3 luciferase constructs ligated with different DAPKl 5' upstream inserts (#1 , #2 , #3 , and #4 were transfected into Jurkat cells and reporter activity was studied 48 hrs after transfection.
  • SV40 promoter ligated to the luciferase reporter was used as a positive control (Ct+) and the pGL3 vector without insert was used as a negative control (Ct-). Luciferase activity for each construct is shown relative to Renilla expression.
  • a schematic of DAPKl upstream region relative to the predicted transcription start site is shown.
  • FIGURE 2B Quantitative DAPKl expression analysis using SYBR green RT-PCR was performed on RNA extracted from normal B-cells and seven CDl 9+ selected CLL samples. The expression is shown relative to one B-cell (defined as 1.0).
  • FIGURE 2C DAPKl expression in 50 CLL samples as measured by semi quantitative RT-PCR and compared to its expression in 6 normal B cells. The distribution is significantly different (p ⁇ 0.01).
  • FIGURE 2D Luciferase assays in 293T cells with either methylated or unmethylated DAPKl construct Wl. Error bars indicate SD.
  • FIGURE 2E Raji cells were treated with 0.5 ⁇ M Decitabine. RT-PCR was performed for DAPKl and GAPDH on untreated and treated cell lines. cDNA synthesized from Jurkat cells was used as a positive control.
  • FIGURE 2F Bisulfite treatment for untreated and 6 and 12 days 5-aza-T- deoxycytidine treated (0.5 ⁇ M) Raji cells.
  • Bisulfite treated DNA was amplified for the BSl region (c.1-1509 to c.1-1262) comprising 30 CpGs and cloned. Sequencing was done for 7-10 clones for each sample. Each row represents a clone. The open circles indicate unmethylated CpG, and closed circles indicate methylated CpG. The overall methylation frequency is given in (%) on the right.
  • FIGURES 3A - 3E show DAPKl regulates apoptosis in lymphoid cells:
  • FIGURES 3 A and 3B -DAPKl expression was studied in two different CLL samples (CLLl and CLL2) cultured with and without HeLA cells for 1 to 6 days as shown. RNA was extracted and RT-PCR was performed on CLL samples. Raji and Jurkat cells were used as negative and positive controls respectively. GAPDH was used as an internal control.
  • FIGURE 3C A heat map of quantitative methylation data for the DAPKl promoter region within amplicon Al in CLLl cells at day 0 and CLLl cells cultured with and without HeLA cells at day 3 as measured by the MassARRAY system. Each line represents a sample and each square an analyzed CpG dinucleotide. Methylation frequencies extend from light yellow (0%) to dark blue (100%).
  • FIGURE 3E Percent live cells were measured in Jurkat cells stably transfected with vector alone or DAPKl siRNA-C, treated with activating Fas antibody (lOOng/ml). After 16 hrs, cells were harvested and suspended in binding buffer with annexin V-FITC and propidium iodide, followed by flow cytometry to assess cell death.
  • FIGURES 4A - 4F show DAPKl expression in CLL family 4532:
  • FIGURE 4A Pedigree of CLL family #4532. Open circle and square represents unaffected female and male, respectively, while closed circle and square represents affected female and male.
  • FIGURE 4B Sequencing of the c.114G>A SNP from genomic DNA and eDNA of two unaffected (IV-2 and IV-4) and two affected (III-3 and III-4) individuals from the family #4532.
  • FIGURE 4C Semi-quantitative SYBRgreen RT-PCR on RNA isolated from monochromosomal hybrid clones with either WT or the CLL chromosome 9 from fibroblast cells of individual 111-4. RPL4 was used as an internal control.
  • FIGURE 4D - DAPKl expression from WT and CLL alleles in monochromosomal hybrids Jurkat and WAC3CD5 cells were used as positive controls (Ct+) and NIH3T3 cells were used as the negative control (Ct-) to show that the antibody used is specific to human DAPKl.
  • FIGURE 4E Allelic expression of DAPKl in affected and unaffected family members.
  • RNA was extracted from one unaffected (IV -3) and one affected (III-4) fibroblast cell line and RT-PCR was performed using primers that amplify the c.l510G>A SNP.
  • the PCR products were cloned and genotyped for either the A or the G allele.
  • FIGURE 4F Allelic variation in DAPKl expression from the two alleles in diploid cells was studied using c.l608C>T SNP as a marker.
  • the A/G ratio in cDNA was normalized to A/G ratio from the genomic DNA.
  • FIGURES 5A - 5F show mutation at c.1-653 lbp regulates DAPKl expression in CLL family:
  • FIGURE 5A - A 357bp PCR product with SNP c.1-6531 A>G was ligated upstream to DAPKl promoter (c.1-2215 to c.1-1151) reporter construct with either A (DAP-A) or G (DAP-G) as SNP.
  • FIGURE 5B The DAPKl promoter alone (#1), DAP-A and DAP-G constructs were transfected into Jurkat cells and the luciferase activity was measured. Renilla expression was used as transfection control.
  • FIGURE 5C Nuclear extracts from Jurkat cells were analyzed by EMSA assay using WT or CLL oligo. Five specific bands are marked.
  • FIGURE 5D For competition EMSA, where indicated, 10 or 50-fold molar excess concentrations of cold oligos relative to the radiolab led oligo were used. Unlabeled Oct-1 oligo was used as a negative control.
  • FIGURE 5E - EMSA assay was performed using WT mutant and CLL mutant oligo where the adjacent bases to the c.1-6531 SNP were mutated.
  • FIGURE 5F For the supershift assay, antibodies against HOXB7, USF2 and MSX2 were added in Jurkat nuclear extract, and gel shift was studied using CLL oligo.
  • FIGURE 5G - DAPKl expression in normal cells and CD 19 selected CLL cells Semi-quantitative RT-PCR was performed on four normal B-cells, three each of normal T- cells and granulocytes and seven selected CLL samples to study expression of DAPKl. GAPDH expression was used as an internal control.
  • FIGURE 5H - HOXB7 expression in normal cells and CD 19 selected CLL cells Semi-quantitative RT-PCR was performed on four normal B-cells, three each of normal T- cells and granulocytes and seven selected CLL samples to study expression of HOXB7. GAPDH expression was used as an internal control.
  • FIGURES 6A - 6E show down-regulation of DAPKl expression by HOXB7 :
  • FIGURES 6A and 6B Fibroblast cell line from affected (III-4) and unaffected (IV-4) family members were transfected with 6OnM of HOXB7 siRNA or scrambled siRNA (Ct-) for different time points and HOXB 7 expression was studied by quantitative SYBR green RT-PCR. HOXB7 expression in untreated cells was set as 1.0.
  • FIGURES 6C and 6D - DAPKl expression was studied in the fibroblasts transfected with H0XB7 siRNA or scrambled siRNA (Ct-) for different time points by quantitative SYBR green RT-PCR.
  • FIGURE 6E Allele specific expression was studied in fibroblasts from III-4 and IV -4 before and after transfection of H0XB7 siRNA. RT-PCR was performed using primers that amplify the het c.1510G>A. The PCR products were cloned and genotyped. The plot shows the ratio between A and the G allele.
  • FIGURES 7A - 7B show DAPKl expression and promoter methylation in CLL cells of family #4532:
  • FIGURE 7 A - shows semi-quantitative RT-PCR for DAPKl with GAPDH as an internal control on RNA extracted from blood cells of #4532 family members III- 1 , 111-2, III-3, III-4 and IV-5 and from separated CD 19+ normal B-cells from 3 healthy volunteers.
  • FIGURE 7B DNA methylation analysis in CLL cells from affected and unaffected family members.
  • Bisulfite treated DNA was amplified for unaffected (IV-I) and affected (III- 1 and III-4) for the BSl and BS2 regions.
  • the PCR products were cloned and sequenced. Each row represents a clone.
  • the open circles indicate unmethylated CpG, and closed circles indicate methylated CpG.
  • FIGURES 8A - 8E show DAPKl promoter methylation and histone tail modifications:
  • FIGURE 8A Schematic of the two regions amplified in ChIP assay, ChIPl (c.l- 1208 to c.l-1151bp) and ChIP2 (c.1-1210 to c.l-1061bp) .
  • DAPKl exon 1 black box
  • the promoter region frequently methylated in CLL samples gray line
  • FIGURE 8B - COBRA assays for two regions in the DAPKl promoter performed on Ramos (Burkitt Lymphoma), BJAB (Atypical Burkitt Lymphoma (EDV-), MEC-I (CLL), MEC-2 (CLL), Was3CD5 (CLL), Daudi (Burkitt Lymphoma) 697 (ALL), RSl 1846 (Non-Hodgkin Lymphoma) RS 4; 11 (ALL) cell line DNAs and CD 19+ selected B-cells.
  • (M) indicates digested PCR products representing the methylated portion and (UM) represents the unmethylated portion.
  • FIGURE 8D Acetylation of histone H3 at lysine 9 and histone H4 are hallmarks of active chromatin.
  • Histone H3 and H4 acetylation status was studied in Raji, WAC3CD5 and Jurkat cell lines within ChIP2 region using anti-acetylated histones H3 and H4 antibodies. No antibody or IgG was used as a negative control.
  • FIGURES 8E and 8EE Quantitative ChIP assay for ChIP 1 region in Raji, Jurkat and WAC3CD5 cell lines using anti acetylated histones H3-K9 and H4 antibody. IgG was used as a negative control.
  • the cell lines expressing DAPKl 5 (Jurkat and WAC3CD5) were enriched for ac-H3-K9 and ac-H4 in DAPKl promoter region, while in Raji cells, the same region lacked the markers of open chromatin.
  • FIGURE 9 Shows cold competition with A oligo and cold competition with G oligo at 1OX and 5OX.
  • FIGURE 10 - shows the sequence information for SEQ ID NOs. 1 - 8.
  • DAPKl death associated protein kinase 1
  • DAPKl expression of the mutated allele is downregulated by 75% in germline cells due to increased H0XB7 binding.
  • promoter methylation results in additional loss of DAPKl expression.
  • reduced expression of DAPKl can act both as a germline predisposing event, and as an epigenetic or genetic somatic event causing or contributing to the CLL phenotype.
  • DAPKl death associated protein kinase 1
  • a mutation in the homeobox containing transcription factor, HOXB7 , binding site in DAPKl regulatory region and/or promoter methylation resulted in significant reduction in the expression of the pro-apoptotic gene DAPKl in familial cases of CLL and in sporadic CLL.
  • DAPKl was initially isolated as a positive mediator of apoptosis induced by interferon gamma (INF-y) (Deiss et al. 5 1995).
  • INF-y interferon gamma
  • FOGURE IA extended DAPKl promoter CpG island
  • DAPKl promoter construct Wl (c.1-1545 to c.1-1151bp) , covering bisulfite region Al (displaying extensive promoter methylation; FIGURE 1), showed a thirty-fold increase in the reporter activity relative to the vector control (FIGURE 2A).
  • FIGURE 2B Semi-quantitative RT-PCR analysis of CD19+ CLL samples used in FIGURE IA, showed reduced DAPKl expression in all seven samples (FIGURE 2B). Furthermore, DAPKl expression in 50 unselected CLL cells also showed statistically significant (p ⁇ 0.01) reduction in all but three CLL samples as compared to normal CD19+ B-cells (FIGURE 2C).
  • FIGURE 2D shows luciferase assays in 293T cells with either methylated or unmethylated DAPKl construct #2. Error bars indicate SD.
  • DAPKl regulates apoptosis in lymphoid cells
  • Jurkat cells with inhibited DAPKl expression showed a statistically significant increase in resistance to apoptosis compared to cells transfected with vector alone (p ⁇ 0.001; FIGURE 3E).
  • CLL was diagnosed in the father (II- 1), his four sons (III- 1 , III-2, III-3, III -4), a grandson (IV-5) and a distant female relative (III-6). Genome-wide linkage analysis was performed using samples III-2, III-3, III-4, IV-3, IV-4 and IV-5 with a panel of 400 microsatellite markers.
  • DAPKl was sequenced from DNA of skin fibroblasts of four affected (III- 2, 111-3, 111-4 and IV-5) as well as two unaffected family members (IV-3 and IV-4) and compared to the genomic sequence (NM_004938) . Although six DNA variants were detected (one in exon 3, two in exon 16, and three in exon 26), none was unique for the CLL haplotype. These variants were useful in subsequent RT-PCR and sequence analysis of DAPKl and showed that gene expression was highly reduced in one allele (CLL allele) in CLL patient samples 111-4 and 111-3 whereas both alleles were expressed in unaffected individuals (FIGURE 4B).
  • Genomic DNA and cDNA from affected and unaffected family members was amplified including SNP C.1608OT.
  • fluorescently-labeled primers specific for either one of the alleles, were used in a primer extension reaction and the intensities were quantified by comparing the ratios of the two alleles in cDNA and gDNA samples.
  • the unaffected individual IV-4 showed a gDNA/cDNA ratio of 0.95 while in the affected individual III-4 the ratio was 0.36 as shown in FIGURE 4F.
  • FIGURE 5E shows that while the mutation in the WT oligo resulted in elimination of band V, mutation of the CLL oligo resulted in elimination of bands FV and V.
  • This result indicated that the WT and the CLL allele might bind similar complexes with different protein components.
  • this result showed that protein binding at band IV is affected by the c.1-6531A>G mutation. Similar results were obtained using Raji nuclear extract (data not shown).
  • EMSA is not a quantitative assay, the inventors observed in multiple independent experiments that the intensity of band IV with the CLL oligo was stronger than with the WT oligo hinting at a differential binding strength (FIGURES 5C, 5D and 5E).
  • HOX family proteins might have differential affinity to A or G basepair at the c.1-6531 SNP.
  • the inventors performed supershift assays using three HOX family proteins (H0XB7 , MSXl and MSX2 ). Only HOXB7 antibody induced a clear supershift, suggesting that H0XB7, or another closely related HOX protein, interacts with this site (FIGURE 5F).
  • DAPKl and HOXB7 expression were tested in B cells, T cells, and granulocytes of three health donors and selected B cells from CLL patients.
  • DAPKl expression was highest in normal B cells, and about one-fifth in granulocytes, respectively (FIGURE 5G).
  • Expression of DAPKl was further reduced (on not detectable) in selected CLL cells.
  • HOXB7 was expressed equally in B and T cells, however much reduced in granulocytes.
  • HOBX7 was variably expressed in selected CD 19+ CLL cells with several samples showing increased expression (FIGURE 5H).
  • DAPKl up regulation in HOXB7 siRNA transfected fibroblasts was observed in III-4 and F/-4, albeit at different levels.
  • DAPKl upregulation was 9-fold while in an unaffected member it was 5-fold compared to the untransfected fibroblast (FIGURES 6C and 6D).
  • allele specific expression analysis by genotyping cloned RT-PCR products showed that activation of the CLL allele was significantly higher than activation of the WT allele supporting the notion that suppression by HOXB7 is higher in the CLL allele than in the WT allele.
  • FIGURE 6E shows that suppression by HOXB7 is higher in the CLL allele than in the WT allele.
  • FIGURE 9 shows cold competition with A oligo and cold competition with G oligo at 1OX and 50X.
  • DAPKl as a tumor suppressor gene in CLL. While linkage studies did not provide sufficient resolution, for the first time the inventors were able to include epigenetic data to pinpoint a candidate gene in a CLL family. The inventors identified a mutation, predisposing to CLL, in the regulatory region of DAPKl in a large family with seven affected individuals. This mutation enhances the binding affinity of transcription factor HOXB7, and results in down-regulation of DAPKl transcription.
  • DAPKl is not only a target in familial CLL, but is also inactivated in the majority of sporadic cases of CLL by epigenetic mechanisms.
  • the inventors' findings provide new insight into the extrinsic and intrinsic pathways of apoptosis, in which DAPKl participates, and highlights the importance of normal DAPKl expression in normal B-cells.
  • this invention demonstrates the need to integrate genetic and epigenetic data for the successful discovery of predisposing genes in cancer.
  • high-penetrance genes e.g. MLHl, BRCAl, pi 6, etc.
  • MLHl MLHl, BRCAl, pi 6, etc.
  • DAPKl DAPKl in CLL
  • Inactivating germline mutations of MLHI cause strong hereditary disposition to the Hereditary Nonpolyposis Colorectal Cancer syndrome.
  • approximately 1% are due to such mutations.
  • additional 12-16% of sporadic colon cancers are due to silencing of MLHJ by acquired promoter hypermethylation (Lynch and de Ia Chapelle, 2003).
  • DAPKl is an actin-filament associated, calcium calmodulin-dependent, serine/threonine kinase that promotes apoptosis in response to various stimuli including Fas, INF-y and TNF-a. (Bialik and Kimchi, 2006). Increased DAPKl expression leads to death-associated cellular alterations and cell morphology changes (Cohen et al., 1997).
  • DAPKl silencing in other human malignancies is mediated by promoter methylation, associated with chromatin changes (Esteller, 2003; Gustafson et al., 2004; Hou et al., 2006; Neuhausen et al., 2006; Toyooka et al., 2003; Yegnasubramanian et al., 2004).
  • the DAPKl promoter contains TGF-f3 response elements as well as p53 binding sites (Martoriati et al., 2005).
  • DAPKl overexpression results in upregulation of p53, suggesting a signaling feedback loop where DAPKl and p53 regulate each other's expression.
  • DAPKl suppresses cMYC and E2F induced cell transformation by activating pl9Al/p53 dependent apoptosis, and also blocks tumor metastasis in vivo (Inbal et al., 1997; Raveh et al., 2001).
  • DAPKl inhibits extracellular signal-regulated kinase (ERK) activity, and counteracts its survival signal (Chen et al., 2005), and promotes cytochrome c release from the mitochondria in response to TGF- 13 induced apoptosis (Jang et al., 2002).
  • ERK extracellular signal-regulated kinase
  • CLL cells express CD40 on their surface and undergo activation upon interaction with CD40 ligand expressing cells, such as HeLa cel]s-(Buhmann et al., 2002).
  • CD40 activation induces expression of several immune accessory molecules, turns CLL cells into antigen presenting cells and also upregulates expression of CD95 (Fas), a member of the tumor necrosis factor (TNF) receptor family, which triggers apoptosis when engaged by the Fas ligand (Dicker et al., 2005).
  • Fas CD95
  • TNF tumor necrosis factor
  • CLL cells cultured with HeLa showed susceptibly to Fas induced apoptosis, upregulation of DAPKl message, and simultaneous demethylation at its promoter suggesting a direct link between CLL cell death and DAPKl expression.
  • Modulating DAPKl expression in Jurkat cells using siRNA provided an additional proof showing its direct involvement in apoptosis.
  • tumor suppressor genes from this region include miR15 and miR16, two microRNAs targeting the oncogene BCL2 (Bullrich et al., 2001; Calin et al., 2002; Cimmino et al., 2005; Hammarsund et al., 2004; Mertens et al., 2002).
  • Other candidate genes include p53 (17pl3) (Lin et al., 2003) and ATM (1 Ig22) (Stankovic et al., 2002).
  • RNA genes (Calin and Croce, 2006; Esquela- Kerscher and Slack, 2006), play important roles in the deregulation of DAPKl, and other crucial genes, contributing to CLL. It was previously reported that such transacting genes show Mendelian inheritance (Morley et al., 2004), which suggests that they may account for a previously demonstrated linkage to multiple loci, however these putative genes need to be explored.
  • HOXB7 is a homeobox containing transcription factor mediating a variety of developmental processes, including hematopoietic differentiation and lymphoid development (LiIl et al, 1995; Shen et al., 1989).
  • DAPKl may at the end of the signaling cascade and modulation of any other upstream members could affect the downstream target, DAPKl.
  • activation of HOXB7 could, occur by activating mutations or loss of upstream repressors regulating this HOXB7.
  • IkappaBalpha an inhibitor of NF-kB activity, has been identified and it was shown that IkappaB-alpha enhances transcriptional activity of HOXB7 by direct interaction (Chariot et al., 1999).
  • An alternative scenario that may explain frequent targeting of DAPKl promoter by aberrant DNA methylation may be the aberrant recruitment of DNA methyltransferase activity to the DAPKl promoter by oncogenic proteins (Brenner et al., 2005; Di Croce et al., 2002).
  • DAPKl as a novel tumor suppressor gene in CLL.
  • the frequent silencing in sporadic CLL shows that aberrant silencing of DAPKl is an early and required event in leukemogenesis.
  • Screening of familial CLL cases for DAPKl promoter methylation can help in early diagnosis of CLL, which is a late onset disease.
  • DNA methylation events are reversible, the inventors' discovery is useful for the development of novel treatment regimens in CLL involving epigenetic therapies for gene reactivation.
  • the skin fibroblasts from family 4532 were cultured in DMEM medium, supplemented with 16% FCS and 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin.
  • Raji cells were treated with Decitabine (5-aza-2'-deoxycytidine) (Sigma- Aldrich, St. Louis MO) at 0.5 ⁇ M concentration for 3, 6, 9 and 12 days.
  • DNA and RNA Extraction DNA and RNA were extracted from the patient samples and cell lines as described previously (Rush et al., 2004).
  • MassARRAY system for Quantitative DNA Methylation Quantitative high- throughput DNA methylation analysis was done as described elsewhere (Ehrich et al., 2005). In brief, the DNA was bisulfite treated using EZ-96 DNA methylation kit (Zymo Research, Canada). Primers designed for PCR did not include CpGs, which allowed amplification of both methylated as well as unmethylated DNA. The primer sequences are available upon request. PCR of bisulfite treated DNA converted uracil to thymidine. The PCR products are then transcribed to RNA and cleaved base specifically. The C/T variation appears as G/A variation in the cleavage product. The G/A variations result in a mass difference of 16Da per CpG site, which is detected by the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALTDI-TOF-MS).
  • MALTDI-TOF-MS matrix-assisted laser desorption
  • RNA samples were used for reverse transcription using SUPERSCRIPTTM First-Strand Synthesis kit (Invitrogen).
  • SYBRgreen PCR was done in triplicates with IQ SYBR Green Supermix (Bio-Rad, Hercules, CA) in a BioRad icycler.
  • the expression data was analyzed by comparative Ct method.
  • Ct represents the cycle number at which the fluorescent signal first exceeds the threshold.
  • the ACt value was obtained by subtracting the Ct value of the internal control (GAPDH or RPL4) from the Ct value of the target gene.
  • the pGL3 basic vector (Promega, Madison, WI) was modified by introducing Notl and EcoRV restriction sites (Yu et al., 2005). These sites were used to ligate the PCR amplified DAPKl promoter constructs of different length.
  • telomeres After two hours l ⁇ g plasmid pGL3 vector and 20ng of pRL-TK internal control vector (Promega) were co-transfected into cells using Polyethylenimine (Polysciences, Warrington, PA). After 4 hours, RPMI media with 10% FCS was added to each well to make up the volume to 1 ml. The cells were further incubated for 48 hours and the luciferase assay was performed according to manufactures instructions (Promega). Luciferase activity was normalized using pRL-TK activity. Each experiment was performed in triplicate.
  • genomic DNA was treated with sodium bisulfite according to published protocols (Herman et al., 1996). Regions BSl and BS2 were amplified (primers are available upon request).
  • the PCR product was purified using the Qiagen Gel Extraction kit (Qiagen, Chatsworth, CA) according to the manufacturer's protocol and subcloned using the TOPO TA-Cloning kit (Invitrogen) followed by sequencing of 8-10 clones for each sample using ABI big dye technology.
  • Chromatin immunoprecipitation (ChIP) assay [00138] Chromatin immunoprecipitation (ChIP) assay:
  • Chromatin immunoprecipitation was carried out using the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY), as described previously (Tada et al., 2006).
  • the antibodies used were rabbit polyclonal anti-acetylated H3-K9, anti-acetylated H3 and anti-acetylated H4 antibodies (Upstate Biotechnology, Lake Placid, NY). Five micrograms of each antibody was used for immunoprecipitation and no antibody or 5ug of rabbit IgG was used as negative controls. SYBR green semiquantitative PCR was performed as described before for the quantitation. Fold difference was calculated for each cell line relative to the negative control rabbit IgG.
  • siRNA transfection For stable transfection of DAPKl siRNA into Jurkat cells, pRS vector with different DAPKl siRNA inserts (siRNA A and C) from OriGene, Rockville, MD were used. Ten micrograms of pRS-DAPKl siRNA A , siRNA C construct or pRS vector alone were transfected into the amphotropic Pheonix packaging cell line (60% confluent) using Superfect (Qiagen). Virus-containing medium was collected from the Pheonix cells after 48hrs, and cell debris was removed by centrifugation.
  • Genomic amplification and SSCP analysis [00148] To screen the control and CLL samples for c.l-6531A>G mutation, genomic DNA was isolated (as described) and amplified using flanking primers. The amplified fragments were analyzed by Single Strand Conformation Polymorphism (SSCP) as previously described (Liechti-Gallati et al., 1999). Variant bands were re-amplified and used for direct sequencing on ABI Prism 3730 DNA analyzer (Applied Biosystems). For genotyping of the alleles for c.l-1510A>G SNP, the cDNA from unaffected III-4 and affected IV -4 samples with and without HOXB7 siRNA transfection were amplified and cloned.
  • SSCP Single Strand Conformation Polymorphism
  • Genomic DNA and cDNA for the marker SNP was amplified using primers flanking the SNP c.l608C>T acid c.4037A>G.
  • the two alleles were distinguished by fluorescently labeled nucleotide of the SNP in both genomic DNA and cDNA, and the peak area was used to determine the ratio of the two alleles. Primers sequences are available on request.
  • Electrophoretic mobility shift assay (EMS A):
  • EMSA was performed as described elsewhere (Chen et al., 2002) with modifications.
  • the oligonucleotides used were
  • the double-stranded oligonucleotides were endlabeled with [ ⁇ 32P] ATP using T4 polynucleotide kinase enzyme (NEB).
  • NEB T4 polynucleotide kinase enzyme
  • the binding reaction was conducted at room temperature for 20min with 5ug of nuclear extract, 40,000 dpm (0.08 to 0.4ng) of radiolabeled oligonucleotide probe, in 5x Ficoll buffer (1OmM Tris (pH 7.5), ImM DTT, ImM EDTA and 4% Ficoll), 250ng of poly(deoxyinosinic-deoxycytidylic acid) in 75mM KCl and double distilled H2O to make the volume to 15ul .
  • Principles of the present invention are directed to the molecular analysis of CLL cells and to providing methods for obtaining information about consistent molecular alterations that advance both the understanding of the basic biology of such cells as well as the clinically relevant aspects of the molecular epidemiology of CLL.
  • laser capture microdissection-derived RNA can be to be used on microarrays and that array hybridization coupled with hierarchical and non-hierarchical analysis methods provide powerful approaches for identifying candidate genes and molecular profiling associated with CCL.
  • Markers according to the present invention may include any nucleic acid sequence or molecule or corresponding polypeptide encoded by the nucleic acid sequence or molecule which demonstrates altered expression (i.e., higher or lower expression) in CLL samples relative to normal samples (i.e., non-cancerous samples).
  • Nucleic acids according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively. See Albert L. Lehninger, PRINCIPLES OF BIOCHEMISTRY, at 793 800 (Worth Pub. 1982). Indeed, the present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glucosylated forms of these bases, and the like.
  • the polymers or oligomers may be heterogeneous or homogeneous in composition, and may be isolated from naturally-occurring sources or may be artificially or synthetically produced.
  • the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single- stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. Oligonucleotide and polynucleotide are included in this definition and relate to two or more nucleic acids in a polynucleotide.
  • Peptide A polymer in which the monomers are alpha amino acids and which are joined together through amide bonds, alternatively referred to as a polypeptide and/or protein.
  • the amino acids may be, for example, the L-optical isomer or the D-optical isomer.
  • Peptides are often two or more amino acid monomers long, and often 4 or more amino acids long, often 5 or more amino acids long, often 10 or more amino acids long, often 15 or more amino acids long, and often 20 or more amino acid monomers long, for example. Standard abbreviations for amino acids are used (e.g., P for proline). These abbreviations are included in Stryer, Biochemistry, Third Ed., 1988, which is incorporated herein by reference in its entirety for all purposes.
  • An array comprises a solid support with peptide or nucleic acid probes attached to said support.
  • Arrays typically comprise a plurality of different nucleic acid or peptide probes that are coupled to a surface of a substrate in different, known locations.
  • These arrays also described as “microarrays” or colloquially “chips” have been generally described in the art, for example, U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186 and Fodor et al., Science, 251:767 777 (1991). Each of which is incorporated by reference in its entirety for all purposes.
  • arrays may generally be produced using mechanical synthesis methods or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase synthesis methods. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261, and 6,040,193 which are incorporated herein by reference in their entirety for all purposes. Although a planar array surface is preferred, the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Pat. Nos.
  • Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of in an all inclusive device, see for example, U.S. Pat. Nos. 5,856,174 and 5,922,591 incorporated in their entirety by reference for all purposes.
  • Gene expression monitoring is a useful way to distinguish between cells that express different phenotypes. For example, cells that are derived from different organs, have different ages, or different physiological states. In one embodiment, gene expression monitoring can distinguish between cancer cells and normal cells, or different types of cancer cells.
  • Expression profile One measurement of cellular constituents that is particularly useful in the present invention is the expression profile.
  • an "expression profile" comprises measurement of the relative abundance of a plurality of cellular constituents. Such measurements may include RNA or protein abundances or activity levels.
  • An expression profile involves providing a pool of target nucleic acid molecules or polypeptides, hybridizing the pool to an array of probes immobilized on predetermined regions of a surface, and quantifying the hybridized nucleic acid molecules or proteins.
  • the expression profile can be a measurement, for example, of the transcriptional state or the translational state of the cell. See U.S. Pat. Nos. 6,040,138, 6,013,449 and 5,800,992, which are hereby incorporated by reference in their entireties for all purposes.
  • Transcriptional state The transcriptional state of a sample includes the identities and relative abundances of the RNA species, especially mRNAs present in the sample. Preferably, a substantial fraction of all constituent RNA species in the sample are measured, but at least a sufficient fraction is measured to characterize the state of the sample.
  • the transcriptional state is the currently preferred aspect of the biological state measured in this invention. It can be conveniently determined by measuring transcript abundances by any of several existing gene expression technologies.
  • Translational state includes the identities and relative abundances of the constituent protein species in the sample. As is known to those of skill in the art, the transcriptional state and translational state are related.
  • the gene expression monitoring system may comprise a nucleic acid probe array (such as those described herein), membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, 5,800,992 which are expressly incorporated herein by reference in their entireties for all purposes.
  • the gene expression monitoring system may be used to facilitate a comparative analysis of expression in different cells or tissues, different subpopulations of the same cells or tissues, different physiological states of the same cells or tissue, different developmental stages of the same cells or tissue, or different cell populations of the same tissue.
  • differentially expressed means that a measurement of a cellular constituent varies in two samples.
  • the cellular constituent can be either upregulated in the experiment relative to the reference or downregulated in the experiment relative to the reference.
  • Differential gene expression can also be used to distinguish between cell types or nucleic acids. See U.S. Pat. No. 5,800,992.
  • nucleic acid samples may contain transcripts of interest.
  • suitable nucleic acid samples may contain nucleic acids derived from the transcripts of interest.
  • a nucleic acid derived from a transcript refers to a nucleic acid for whose synthesis the mRNA transcript or a subsequence thereof has ultimately served as a template.
  • a cDNA reverse transcribed from a transcript, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc. are all derived from the transcript and detection of such derived products is indicative of the presence and/or abundance of the original transcript in a sample.
  • suitable samples include, but are not limited to, transcripts of the gene or genes, cDNA reverse transcribed from the transcript, cRNA transcribed from the cDNA, DNA amplified from the genes, RNA transcribed from amplified DNA, and the like.
  • Transcripts may include, but are not limited to pre-mRNA nascent transcript(s), transcript processing intermediates, mature mRNA(s) and degradation products. It is not necessary to monitor all types of transcripts to practice this invention. For example, one may choose to practice the invention to measure the mature mRNA levels only.
  • a sample is a homogenate of cells (e.g., blood cells), tissues or other biological samples.
  • a nucleic acid preparation e.g., a total RNA preparation of a biological sample.
  • a nucleic acid sample is the total mRNA isolated from a biological sample.
  • the total mRNA prepared with most methods includes not only the mature mRNA, but also the RNA processing intermediates and nascent pre- mRNA transcripts.
  • total mRNA purified with a poly (T) column contains RNA molecules with poly (A) tails. Those poly A+ RNA molecules could be mature mRNA, RNA processing intermediates, nascent transcripts or degradation intermediates.
  • Biological samples may be of any biological tissue or fluid or cells. Frequently the sample will be a "clinical sample” which is a sample derived from a patient. Clinical samples provide rich sources of information regarding the various states of genetic network or gene expression. Some embodiments of the invention are employed to detect mutations and to identify the function of mutations. Such embodiments have extensive applications in clinical diagnostics and clinical studies. Typical clinical samples include, but are not limited to, sputum, blood, blood cells (e.g., white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.
  • the level of expression of a marker for CLL is assessed by detecting the presence of a nucleic acid corresponding to the marker in the sample.
  • the level of expression of a marker for CLL is assessed by detecting the presence of a protein corresponding to the marker in the sample.
  • the presence of the protein is detected using a reagent which specifically binds to the protein, e.g., an antibody, an antibody derivative, and/or an antibody fragment.
  • Detection can involve contacting a sample with a compound or an agent capable of detecting a marker associated with oral cancer such that the presence of the marker is detected in the biological sample.
  • a compound or an agent capable of detecting marker RNA can be a labeled or labelable nucleic acid probe capable of hybridizing to marker RNA.
  • the nucleic acid probe can be, for example, complementary to any of the nucleic acid markers of oral cancer disclosed herein, or a portion thereof, such as an oligonucleotide which specifically hybridizes marker RNA.
  • One useful agent for detecting a marker protein is a labeled or labelable antibody capable of binding to the marker protein.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, antibody derivative, or a fragment thereof (e.g., Fab or F(ab').sub.2) can be used.
  • the term "labeled or labelable", with regard to the probe or antibody is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Non-limiting examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • the detection methods described herein can be used to detect marker RNA or marker protein in a biological sample in vitro as well as in vivo.
  • In vitro techniques for detection of marker RNA include, but are not limited to, Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of marker protein include, but are not limited to, enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence assays.
  • marker protein can be detected in vivo in a subject by introducing into the subject a labeled antibody against the marker protein.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • RNase present in homogenates before homogenates can be used for hybridization.
  • Methods of inhibiting or destroying nucleases are well known in the art.
  • cells or tissues are homogenized in the presence of chaotropic agents to inhibit nuclease.
  • RNases are inhibited or destroyed by heat treatment followed by proteinase treatment.
  • total RNA is isolated from a given sample using, for example, an acid guanidinium-phenol-chloroform extraction method followed by polyA+ mRNA isolation by oligo dT column chromatography or by using (dT)n magnetic beads (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), VoIs. 1 3, Cold Spring Harbor Laboratory, (1989), or Current Protocols in Molecular Biology, F. Ausubel et al., ed. Greene Publishing and Wiley-Interscience, New York (1987) each hereby incorporated by reference in their entireties for all purposes). See also PCT/US99/25200 for complexity management and other sample preparation techniques, which is hereby incorporated by reference in its entirety for all purposes.
  • nucleic acid sample it is desirable to amplify the nucleic acid sample prior to hybridization.
  • methods of amplifying nucleic acids are well known in the art and that whatever amplification method is used, if a quantitative result is desired, care must be taken to use a method that maintains or controls for the relative frequencies of the amplified nucleic acids to achieve quantitative amplification.
  • Methods of "quantitative" amplification are well known to those of skill in the art.
  • quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. A high density array may then be performed which includes probes specific to the internal standard for quantification of the amplified nucleic acid.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • RNA lysates or tissue homogenates often contain a number of regulators of polymerase activity. Therefore, the skilled practitioner typically incorporates preliminary steps to isolate total RNA or mRNA for subsequent use as an amplification template.
  • One tube mRNA capture methods may be used to prepare poly(A)+ RNA samples suitable for immediate RT-PCR in the same tube (Boehringer Mannheim). The captured mRNA can be directly subjected to RT-PCR by adding a reverse transcription mix and, subsequently, a PCR mix.
  • the sample mRNA is reverse transcribed with a reverse transcriptase and a primer consisting of oligo dT and a sequence encoding the phage T7 promoter to provide single stranded DNA template.
  • the second DNA strand is polymerized using a DNA polymerase.
  • T7 RNA polymerase is added and RNA is transcribed from the cDNA template. Successive rounds of transcription from each single cDNA template results in amplified RNA.
  • Methods of in vitro polymerization are well known to those of skill in the art (see, e.g., Sambrook, supra).
  • aRNA antisense RNA
  • the oligonucleotide probes provided in the array are chosen to be complementary to subsequences of the antisense nucleic acids.
  • the target nucleic acid pool is a pool of sense nucleic acids
  • the oligonucleotide probes are selected to be complementary to subsequences of the sense nucleic acids.
  • the probes may be of either sense as the target nucleic acids include both sense and antisense strands.
  • the protocols cited above include methods of generating pools of either sense or antisense nucleic acids. Indeed, one approach can be used to generate either sense or antisense nucleic acids as desired.
  • the cDNA can be directionally cloned into a vector (e.g., Stratagene's p Bluscript II KS (+) phagemid) such that it is flanked by the T3 and T7 promoters. In vitro transcription with the T3 polymerase will produce RNA of one sense (the sense depending on the orientation of the insert), while in vitro transcription with the T7 polymerase will produce RNA having the opposite sense.
  • a vector e.g., Stratagene's p Bluscript II KS (+) phagemid
  • In vitro transcription with the T3 polymerase will produce RNA of one sense (the sense depending on the orientation of the insert), while in vitro transcription with the T7 polymerase will produce RNA having the opposite sense.
  • Other suitable cloning systems include phage lambda
  • laser dissection microscopy is one method that can be used in the present invention.
  • Other techniques include L. Zhang et al., Science 276, 1268 (1997), Mahadevappa, M. & Wa ⁇ ington, J. A. Nat. Biotechnol. 17, 1134 1136 (1999) and Luo, L. et al. Nature Med. 5, 117 122 (1999) which are all hereby incorporated by reference in their entireties for all purposes.
  • the invention provides methods of assessing the efficacy of test compounds and compositions for treating CLL.
  • the methods include identifying candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which have an inhibitory effect on CLL.
  • candidate or test compounds or agents e.g., peptides, peptidomimetics, small molecules or other drugs
  • Candidate or test compounds or agents which have an inhibitory effect on CLL are identified in assays that employ CLL cancer cells, such as an expression assay entailing direct or indirect measurement of the expression of a CLL marker (e.g., a nucleic acid marker or a protein marker).
  • a CLL marker e.g., a nucleic acid marker or a protein marker
  • modulators of expression of CLL markers can be identified in a method in which a cell is contacted with a candidate compound and the expression of CLL markers (e.g., nucleic acid markers and/or protein markers) in the cell is determined.
  • CLL markers e.g., nucleic acid markers and/or protein markers
  • the level of expression of CLL markers in the presence of the candidate compound is compared to the level of expression of CLL markers in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of expression of CLL based on this comparison.
  • kits for assessing whether a subject is afflicted with CLL as well as kits for assessing the presence of CLL cells.
  • the kit may comprise a labeled compound or agent capable of detecting CLL markers (e.g., nucleic acid markers and/or protein markers) in a biological sample, a means for determining the amount of CLL markers in the sample, and a means for comparing the amount of CLL markers in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect CLL markers.
  • a relational database is preferred and can be used, but one of skill in the art will recognize that other databases could be used.
  • a relational database is a set of tables containing data fitted into predefined categories. Each table, or relation, contains one or more data categories in columns. Each row contains a unique instance of data for the categories defined by the columns.
  • a typical database for the invention would include a table that describes a sample with columns for age, gender, reproductive status, expression profile and so forth. Another table would describe a disease: symptoms, level, sample identification, expression profile and so forth.
  • the invention matches the experimental sample to a database of reference samples.
  • the database is assembled with a plurality of different samples to be used as reference samples.
  • An individual reference sample in one embodiment will be obtained from a patient during a visit to a medical professional.
  • the sample could be, for example, a tissue, blood, urine, feces or saliva sample.
  • Information about the physiological, disease and/or pharmacological status of the sample will also be obtained through any method available. This may include, but is not limited to, expression profile analysis, clinical analysis, medical history and/or patient interview. For example, the patient could be interviewed to determine age, sex, ethnic origin, symptoms or past diagnosis of disease, and the identity of any therapies the patient is currently undergoing. A plurality of these reference samples will be taken.
  • a single individual may contribute a single reference sample or more than one sample over time.
  • confidence levels in predictions based on comparison to a database increase as the number of reference samples in the database increases.
  • some of the indicators of status will be determined by less precise means, for example information obtained from a patient interview is limited by the subjective interpretation of the patient. Additionally, a patient may lie about age or lack sufficient information to provide accurate information about ethnic or other information. Descriptions of the severity of disease symptoms is a particularly subjective and unreliable indicator of disease status.
  • the database is organized into groups of reference samples. Each reference sample contains information about physiological, pharmacological and/or disease status.
  • the database is a relational database with data organized in three data tables, one where the samples are grouped primarily by physiological status, one where the samples are grouped primarily by disease status, and one where the samples are grouped primarily by pharmacological status. Within each table the samples can be further grouped according to the two remaining categories. For example, the physiological status table could be further categorized according to disease and pharmacological status.
  • the present invention may be embodied as a method, data processing system or program products. Accordingly, the present invention may take the form of data analysis systems, methods, analysis software, and the like.
  • Software written according to the present invention is to be stored in some form of computer readable medium, such as memory, hard-drive, DVD ROM or CD ROM, or transmitted over a network, and executed by a processor.
  • the present invention also provides a computer system for analyzing physiological states, levels of disease states and/or therapeutic efficacy.
  • the computer system comprises a processor, and memory coupled to said processor which encodes one or more programs.
  • U.S. Pat. No. 5,733,729 illustrates an example of a computer system that may be used to execute the software of an embodiment of the invention.
  • This patent shows a computer system that includes a display, screen, cabinet, keyboard, and mouse.
  • the mouse may have one or more buttons for interacting with a graphic user interface.
  • the cabinet preferably houses a CD-ROM or DVD-ROM drive, system memory and a hard drive which may be utilized to store and retrieve software programs incorporating computer code that implements the invention, data for use with the invention and the like.
  • a CD is shown as an exemplary computer readable medium, other computer readable storage media including a floppy disk, a tape, a flash memory, a system memory, and a hard drive may be utilized.
  • a data signal embodied in a carrier wave may be the computer readable storage medium.
  • the patent also shows a system block diagram of a computer system used to execute the software of an embodiment of the invention.
  • the computer system includes a monitor, a keyboard, and a mouse.
  • the computer system further includes subsystems such as a central processor, a system memory, a fixed storage (e.g., a hard drive), a removable storage (e.g., CD-ROM), a display adapter, a sound card, speakers, and a network interface.
  • Other computer systems suitable for use with the invention may include additional or fewer subsystems.
  • another computer system may include more than one processor or a cache memory.
  • Computer systems suitable for use with the invention may also be embedded in a measurement instrument.
  • the embedded systems may control the operation of, for example, a GENECHIP.RTM. Probe array scanner (also called a GENEARRAY.RTM. scanner sold by Agilent corporation, Palo Alto, Calif.) as well as executing computer codes of the invention.
  • Computer methods can be used to measure the variables and to match samples to eliminate gene expression differences that are a result of differences that are not of interest. For example, a plurality of values can be input into computer code for one or more physiological, pharmacological and/or disease states. The computer code can thereafter measure the differences or similarities between the values to eliminate changes not attributable to a value of interest.
  • Computer software to analyze data generated by microarrays is commercially available from Affymetrix Inc. (Santa Clara) as well as other companies.
  • Affymetrix Inc. distributes GENECHIP.RTM., now known as MicroArray suite, LIMS, Microdb, Jaguar, DMT, and other software.
  • Other databases can be constructed using the standard database tools available from Microsoft (e.g., Excel and Access).
  • High-density oligonucleotide arrays are particularly useful for monitoring the gene expression pattern of a sample.
  • total mRNA isolated from the sample is converted to labeled cRNA and then hybridized to an array such as a GENECHIP.RTM. oligonucleotide array.
  • Each sample is hybridized to a separate array. Relative transcript levels are calculated by reference to appropriate controls present on the array and in the sample.
  • CD40L stimulation enhances the ability of conventional metaphase cytogenetics to detect chromosome aberrations in B- cell chronic lymphocytic leukaemia cells. Br J Haematol 118, 968-975.
  • ZAP-70 methylation status is associated with ZAP-70 expression status in chronic lymphocytic leukemia.
  • Haematologica 90, 1078-1088. Dao-Ung, L. P., Fuller, S. J., Sluyter, R., SkarRatt, K. K., Thunberg, U., Tobin, G., Byth,
  • TGF-beta induces apoptosis through Smad-mediated expression of DAP-kinase.
  • DAP kinase Death-associated protein kinase alteration in soft tissue leiomyosarcoma: Promoter methylation or homozygous deletion is associated with a loss of DAP kinase expression.
  • EBV can immortalize B-cll cells activated by cytokines.
  • ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile.

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Abstract

La présente invention concerne un procédé permettant la détermination de la susceptibilité à la leucémie lymphocytique chronique chez un sujet comprenant la détermination d'une perte ou d'une expression réduite de la protéine kinase 1 associée à l'apoptose (DAPK1) ou de fragments ou d'équivalents de celle-ci.
PCT/US2008/002625 2007-03-02 2008-02-28 Protéine kinase 1 associée à l'apoptose (dapk1) et ses utilisations pour le traitement de la leucémie lymphocytique chronique WO2008108964A2 (fr)

Priority Applications (2)

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US9149523B2 (en) 2010-12-15 2015-10-06 Yeda Research And Development Co. Ltd. Compositions and methods for treating cancer and neurodegenerative diseases

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